The present application proposes a concerted biochemical, biophysical, and genetic approach to identifying novel targets in the signaling pathway activated by the EWSR1-FLI1 fusion protein, coupled with two focused high throughput screening campaigns to identify novel compounds selectively active against Ewing sarcoma tumors. In order to fully validate the therapeutic potential of this approach, a well-considered scheme to optimize the ADME properties of any identified hits and test them in both proof-of concept subcutaneous and more rigorous orthotopic and patient-derived xenograft models is necessary. The inclusion of a Preclinical Pharmacology (PK/PD) core is therefore critical to ensure the translational success of our proposal. In Aim 1, we will validate that loss of EWSR1-FLI1 in Ewing tumor lines in vivo using an auxin-based degron system, as outlined in Project 1, results in inhibition of tumor growth. Aim 2 will optimize the absorption, distribution, metabolism and excretion (ADME) properties of inhibitors of EWSR1-FLI1 function, identified in screening campaigns conducted primarily in Project 3 but also from Project 2 if such inhibitors show promising cell-based activity. Aim 3 determines the efficacy of inhibitors of EWRS1-FLI1 function in subcutaneous models of Ewing sarcoma (Project 3). Compounds will be tested in two separate Ewing sarcoma cell-based models as well as one control rhabdomyosarcoma. Finally, Aim 4 will develop an orthotopic model of Ewing sarcoma in order to test the most promising inhibitors of EWSR1-FLI1 function and the effects of in vivo loss of EWSR1-FLI in the auxin-degron model (Projects 1 and 3). Specific and distinct interactions with bone are central to the pathogenesis of Ewing sarcoma and it will be important to distinguish whether our newly identified inhibitors function in this context.